Mapping 250K/500K SNP assay

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Pipette tips As needed for pipettes listed in Table A.6. 234 GeneChip ® Mapping 500K 96-Well Plate Protocol Table A.9 Other Consumables Required for the Mapping 500K 96-Well Plate Protocol Item Manufacturer/ Distributor Rainin GP-L10F GP-L200F GP-L1000F RT-L10F RT-L200F RT-L1000F GP = refill RT = with rack Pre-PCR and Main Lab Plate seals – see Table A.7 on page 232 Pre-PCR and Main Lab Plates, 96-well PCR – see Table A.7 on page 232 Pre-PCR and Main Lab Plates, 96-well UV Star, 370 µL/well E&K Scientific 25801 Main Lab Solution Basin, 100 mL sterile, multichannel Solution Basin, 55 mL sterile, multichannel Labcor 730-014 Main Lab Labcor 730-004 Pre-PCR and Main Lab Solution Basin lid, 55 mL Labcor 730-021 Pre-PCR and Main Lab Tough-Spots ® Tubes, strip of 12, thin wall (0.2 mL) Diversified Biotech SPOT-1000 USA Scientific 9185-1000 CLP Direct 3426.12 ISC BioExpress T-3114-1 Main Lab Tube, centrifuge 15 mL VWR 20171-020 Main Lab Tube, centrifuge 50 mL VWR 21008-178 Main Lab Tube, Eppendorf 2.0 mL VWR 20901-540 Pre-PCR Tube, Eppendorf Safe-Lock, 1.5 mL (required only if using tubes for Stage 9: Target Hybridization) Part Number Laboratory Location VWR 21008-959 Main Lab Tube, Falcon, 50 mL VWR 21008-940 Pre-PCR Pre-PCR and Main Lab
Supplier Contact List Table A.10 Supplier Contact List Supplier Web Site appendix A | Reagents, Equipment, and Consumables Required for 96-Well Plate Protocol 235 Affymetrix www.affymetrix.com Ambion www.ambion.com Applied Biosystems home.appliedbiosystems.com Bionexus Inc. www.bionexus.net Bio-Rad www.bio-rad.com Boekel Scientific www.boekelsci.com Cambrex www.cambrex.com CLP Direct www.clpdirect.com Clontech www.clontech.com/clontech Diversified Biotech www.divbio.com E&K Scientific www.eandkscientific.com Eppendorf www.eppendorf.com Fisher Scientific www.fisherscientific.com Invitrogen Life Technologies www.invitrogen.com ISC BioExpress www.bioexpress.com Labcor www.labcorproducts.com Molecular Devices www.moleculardevices.com New England Biolabs www.neb.com Pierce Biotechnology www.piercenet.com Promega www.promega.com QIAGEN www.qiagen.com Rainin www.rainin.com Sigma-Aldrich www.sigma-aldrich.com
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GeneChip ® Mapping 500K Assay Manu
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Contents CHAPTER 1 Overview 1 ABOUT
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contents v Equipment and Consumable
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contents vii Reagents Required 118
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contents ix CHAPTER 8 Troubleshooti
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contents xi STEP 4: PCR 257 Reagent
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Chapter 1 Overview
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About This Manual This manual is a
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chapter 1 | Overview 5 LD patterns
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Figure 1.1 GeneChip ® Mapping Assa
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chapter 1 | Overview 9 Gilman, B.,
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chapter 1 | Overview 11 23. Schaid,
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chapter 1 | Overview 13 35. Hu, N.,
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chapter 1 | Overview 15 50. Bignell
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chapter 1 | Overview 17 64. Gabriel
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Chapter 2 Laboratory Setup
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Introduction to Laboratory Setup Pl
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Main Lab Pre-PCR Clean Room chapter
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Chapter 3 Genomic DNA General Requi
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Introduction This chapter describes
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Sources of Human Genomic DNA chapte
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chapter 3 | Genomic DNA General Req
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Chapter 4 96-Well Plate Protocol
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ABOUT THIS PROTOCOL 96-Well Plate P
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chapter 4 | 96-Well Plate Protocol
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PREPARING THE WORK AREA FOR EACH ST
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PROGRAM YOUR THERMAL CYCLERS chapte
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EQUIPMENT AND CONSUMABLES REQUIRED
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ALIQUOTING PREPARED GENOMIC DNA To
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Negative Controls chapter 4 | 96-We
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Table 4.7 Nsp I Digestion Master Mi
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Stage 3: Ligation ABOUT THIS STAGE
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REAGENTS REQUIRED chapter 4 | 96-We
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DILUTE THE SAMPLES To dilute the sa
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Stage 4: PCR ABOUT THIS STAGE chapt
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About Controls chapter 4 | 96-Well
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B P1 P2 P3 Figure 4.2 Transferring
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Table 4.16 PCR Master Mix ADD PCR M
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WHAT YOU CAN DO NEXT chapter 4 | 96
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POOL THE PCR PRODUCTS chapter 4 | 9
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Figure 4.5 Loading Optical Plates w
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Process Control Metrics Evaluate th
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Table 4.24 PROBLEM: Average Sample
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IMPORTANT INFORMATION ABOUT THIS ST
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Figure 4.6 Work Area Set Up for Sta
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Stage 8: Labeling ABOUT THIS STAGE
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LOCATION AND DURATION • Main Lab
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Hybridizing Samples Using Heat Bloc
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Table 4.37 Hybridization Master Mix
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Load the Samples onto Arrays chapte
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Figure 4.8 Applying Tough-Spots ®
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Chapter 5 Washing, Staining, and Sc
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Introduction This chapter contains
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Reagent Preparation Wash A: Non-Str
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Experiment and Fluidics Station Set
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chapter 5 | Washing, Staining, and
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HANDLING THE GENECHIP ® PROBE ARRA
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Chapter 6 Fluidics Station Care and
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Introduction This chapter provides
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chapter 6 | Fluidics Station Care a
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White Clamp Figure 6.7 Releasing pe
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Figure 6.8 Troubleshooting decision
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PROBLEMS AND SOLUTIONS Table 6.3 Co
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Table 6.3 (Continued) Common error
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Table 6.4 (Continued) Common Error
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Table 6.5 (Continued) Other Problem
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Chapter 7 Analysis Workflow
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Introduction Software Requirements
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Page 197 and 198: Figure 7.3 Dynamic Model Mapping Al
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Page 201 and 202: FILE SETS chapter 7 | Analysis Work
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Page 205 and 206: NETAFFX SNP ANNOTATION chapter 7 |
Page 207 and 208: CALL RATE chapter 7 | Analysis Work
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Page 211 and 212: chapter 7 | Analysis Workflow 199 F
Page 213 and 214: chapter 7 | Analysis Workflow 201 S
Page 215 and 216: B2 OLIGO PERFORMANCE chapter 7 | An
Page 217 and 218: chapter 7 | Analysis Workflow 205 3
Page 219 and 220: chapter 7 | Analysis Workflow 207 W
Page 221 and 222: Chapter 8 Troubleshooting
Page 223 and 224: Assay Recommendations 211 Genotypin
Page 225 and 226: chapter 8 | Troubleshooting 213 10.
Page 227 and 228: Problem Likely Cause Solution Faint
Page 229 and 230: Problem Likely Cause Solution Posit
Page 231 and 232: Table 8.2 PROBLEM: Average Sample O
Page 233 and 234: When to Contact Technical Support c
Page 235 and 236: Appendix A Reagents, Equipment, and
Page 237 and 238: Reagents, Equipment, and Consumable
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Page 245: Consumables Required GENECHIP ® AR
Page 249 and 250: Appendix B Thermal Cycler Programs
Page 251 and 252: ABOUT THIS APPENDIX 500K DIGEST 500
Page 253 and 254: 500K FRAGMENT 500K LABEL 500K HYB a
Page 255 and 256: Appendix C Low Throughput Protocol
Page 257 and 258: Introduction The Affymetrix GeneChi
Page 259 and 260: BEFORE YOU BEGIN appendix C | Low T
Page 261 and 262: STEP 2: Restriction Enzyme Digestio
Page 263 and 264: DIGESTION PROCEDURE PRE-PCR CLEAN A
Page 265 and 266: STEP 3: Ligation REAGENTS AND EQUIP
Page 267 and 268: PCR STAGING AREA appendix C | Low T
Page 269 and 270: STEP 4: PCR REAGENTS AND EQUIPMENT
Page 271 and 272: PCR PROCEDURE PRE-PCR CLEAN ROOM ap
Page 273 and 274: MAIN LAB appendix C | Low Throughpu
Page 275 and 276: STEP 5: PCR Purification and Elutio
Page 277 and 278: appendix C | Low Throughput Protoco
Page 279 and 280: STEP 7: Fragmentation REAGENTS AND
Page 281 and 282: FRAGMENTATION PROCEDURE 1. Pre-heat
Page 287 and 288: LABELING PROCEDURE MAIN LAB appendi
Page 289 and 290: STEP 9: Target Hybridization REAGEN
Page 291 and 292: HYBRIDIZATION PROCEDURE appendix C
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Appendix D
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appendix D | Reagents, Instruments,
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* Adaptor, Nsp 5' ATTATGAGCACGACAGA
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Table D.4 Reagents Supplied by Affy
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Fragmentation and Labeling appendix
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Mapping 250K/500K SNP assay

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